Neuropeptide Y (NPY) is a 36 residue C-terminally amidated polypeptide neurotransmitter which is evolutionarily well conserved among mammals. NPY is also the most abundant peptide in the mammalian brain and is found in many central and peripheral neurons, largely co-localized with catecholamines. NPY is anatomically co-distributed and co-released with norepinephrine in and from sympathetic postganglionic neurons. NPY possesses potent vasoconstrictor properties, stimulates food intake and body fat stores, reduces heart rate, increases blood pressure, mediates analgesia, regulates memory processing, and is implicated in the pathology of various disease states, including hypertension, congestive heart failure, obesity and various psychiatric disorders. NPY can act pre-synaptically to inhibit its own release and that of catecholamines, or can potentiate the vasoconstrictor action of other neurotransmitters and hormones such as noradrenaline, angiotensin II and histamine.
NPY exerts its physiological effects by acting on specific biological receptors. Specific NPY binding sites exist in two distinct receptor subtypes, i.e., NPY receptor Y1 and NPY receptor Y2. Postsynaptic NPY receptors are of Y1 and Y2 types, whereas presynaptic receptors are mainly of the Y2 type. Subtype Y1 receptors exist in the sympathetic nervous system predominantly post-synaptically to mediate vasoconstriction and appetite stimulation. Y2 receptors, which are predominant in the central nervous system, act pre-synaptically in the regulation of catecholamine release. An extremely important action of NPY Y2 activation is to decrease cardiac contractility (inotropy). Under many circumstances in which inotropy is decreased, diseases of life-threatening importance, e.g. congestive heart failure and cardiogenic shock, are associated with increased release of NPY into the blood. Use of a selective NPY Y1 agonist to treat hypotension and various forms of shock while avoiding further NPY release is considered to be clinically beneficial in cardiac disorders.
Functional Y1 receptors have been identified on the neuroblastoma cell line SK-N-MC (Gordon, E. A. et al., J. Neurochem., 55:506-513). Functional Y2 receptors have been identified on the cell line SK-N-BE2. At the intracellular level NPY stimulation of Y2 receptors inhibits the activity of adenylate cyclase. Vascular smooth muscle contraction and centrally evoked food intake (appetite stimulation) are predominantly mediated by NPY Y1 receptors.
Distinct structure-activity relationships exist for NPY binding to different biological receptors. The distinction between the two types of receptors is derived from differential binding properties of NPY C-terminal fragments and various modifications of the peptide. Sheikh, S. P., et al. first characterized the Y1 and Y2 NPY-binding sites (FEBS 245:209-214 (1989)). It was reported that, unlike intact NPY, the NPY.sup.13-36 C-terminal fragment binds Y2 but not Y1. Synthesis of NPY analogs with modified N- or C-termini or D-substitutions was further performed to determine the domains responsible for biological activities of the separate receptors (Boublic, J. H., et al., J. Med. Chem., 32:597-601 (1989)). An otherwise intact NPY peptide having substituted amino acids Leu.sup.31 and Pro.sup.34 was shown to be Y1 selective (Fuhlendorff, J., et al., PNAS 87:182-186 (1990)). Several cyclic and centrally truncated analogs of NPY have been reported that show selectivity for Y2 receptors (Beck-Sickinger, A. G., et al., Eur. J. Biochem., 206:957-964 (1992); Kirby, D. A., et al., J. Med. Chem., 36:385-393 (1993)). Improvement of Y1 binding affinity has previously been achieved only by including more residues in the central polypeptide fold (PP-fold) region, while the length of an intramolecular bridge has not previously been shown to affect binding.
Accordingly, there remains a need for stable and potent NPY analogs capable of binding and activating NPY Y1 receptors without substantially binding and activating NPY Y2 receptors in order to mediate appetite stimulation, vasoconstriction, and to detect and localize neuroblastoma tumors without affecting catecholamine release or otherwise propagating physiological effects associated with the activation of Y2 receptors.